本文選題：白介素-10 + 神經炎癥　； 參考：《蘇州大學》2016年博士論文

【摘要】：目的:白介素-10(interleukin-10,IL-10)是最重要和最有效的抗炎細胞因子之一。中樞神經系統(tǒng)中有多種細胞可產生IL-10或表達IL-10受體,這提示IL-10可以通過作用于腦內相關細胞調節(jié)神經炎癥反應。而研究表明帕金森病與神經炎癥之間存在重要關聯(lián)。另外,IL-10也存在抗凋亡作用,那么我們推測IL-10是否可以其抗炎和抗凋亡作用對帕金森病進行防治。因此本研究觀察IL-10在PD發(fā)生發(fā)展過程中對神經炎癥和多巴胺(dopamine,DA)能神經元缺失的影響,并對其作用機制和信號通路進行初步探討,以明確IL-10對DA能神經元的保護作用,從而發(fā)掘IL-10的治療潛力,為防治PD提供實驗依據。方法:1.細胞培養(yǎng):(1)中腦腹側細胞培養(yǎng):取大鼠胚胎14±0.5天的中腦腹側組織進行原代細胞培養(yǎng),培養(yǎng)7天后備用。(2)中腦腹側單純神經元培養(yǎng):取大鼠胚胎14±0.5天的中腦腹側組織進行原代細胞培養(yǎng),用阿糖胞苷抑制膠質細胞生長以獲得神經元,培養(yǎng)7天后備用。(3)中腦腹側神經元-小膠質細胞共培養(yǎng):取新生大鼠大腦皮層組織進行原代細胞培養(yǎng),12-14天后獲取小膠質細胞加入已培養(yǎng)6天的神經元,24 h后備用。(4)中腦腹側神經元-星形膠質細胞共培養(yǎng):取新生大鼠大腦皮層組織進行原代細胞培養(yǎng),12-14天后去除小膠質細胞,至少4次傳代獲取星形膠質細胞加入已培養(yǎng)6天的神經元,24 h后備用。2.上述四種培養(yǎng)體系中,IL-10抑制LPS誘導的神經炎癥和DA能神經元缺失的檢測:用IL-10(15,50或150 ng/m L)預處理培養(yǎng)物1 h后再加入LPS(50 ng/m L)作用8 h(檢測凋亡相關蛋白表達)或24 h。應用免疫熒光細胞化學法檢測DA能和非DA能神經元的數(shù)目,PCR和Western blot方法檢測培養(yǎng)物中炎癥介質(i NOS、COX-2、IL-1β和TNF-α)和神經營養(yǎng)因子(BDNF、IGF-1和GDNF)的水平,ELISA方法檢測培養(yǎng)上清中IL-1β、TNF-α或IGF-1的濃度。另外,中腦腹側細胞培養(yǎng)體系中,應用化學比色法檢測上清中NO和H2O2的濃度;中腦腹側單純神經元培養(yǎng)體系中,應用免疫熒光雙標法觀察LPS受體TLR-4在神經元(Neu N+)上的定位情況,Western blot方法檢測培養(yǎng)物中促凋亡酶(活化的caspase-3和caspase-9)的蛋白表達,原位末端標記法檢測神經元凋亡率。3.中腦腹側細胞培養(yǎng)體系中,IL-10抑制MPP+誘導的神經炎癥和DA能神經元缺失的檢測:用IL-10(15或50 ng/m L)預處理培養(yǎng)物1 h后再加入MPP+(5μM)作用24 h。應用免疫熒光細胞化學法檢測DA能神經元的數(shù)目;Western blot方法檢測培養(yǎng)物中炎癥介質(i NOS、COX-2、IL-1β和TNF-α)和神經營養(yǎng)因子(BDNF、IGF-1和GDNF)的表達;ELISA方法檢測培養(yǎng)上清中IL-1β、TNF-α和IGF-1的濃度。4.中腦腹側單純神經元培養(yǎng)體系中,IL-10抑制MPP+誘導的神經炎癥和DA能神經元缺失的檢測:用IL-10(15或50 ng/m L)預處理培養(yǎng)物1 h后再加入MPP+(5μM)作用8h或24h。應用免疫熒光細胞化學法檢測DA能神經元的數(shù)目;Western blot方法檢測培養(yǎng)物中活化的caspase-3和caspase-9的表達。我們設想IL-10可通過減少TNF-α,抑制細胞凋亡,減輕DA能神經元缺失,于是應用免疫熒光雙標法確定TNF-α在受損DA能神經元的定位情況;Western blot方法檢測培養(yǎng)物TNF-α的蛋白表達;ELISA方法檢測培養(yǎng)上清中TNF-α的濃度。為確認TNF-α在MPP+誘導的神經炎癥中的作用,我們進一步用中和性TNF-α抗體(1μg/m L)預處理培養(yǎng)物2h后,加或不加IL-10(50 ng/m L)處理1 h后,再加入MPP+(5μM)作用8 h或24 h。應用免疫熒光細胞化學法檢測DA能神經元的數(shù)目;Western blot方法檢測培養(yǎng)物中活化的caspase-3和caspase-9的表達。5.中腦腹側單純神經元培養(yǎng)體系中,IL-10通過神經元上IL-10受體和神經元內JAK-STAT3信號通路抑制LPS誘導的神經炎癥和神經元凋亡的檢測:首先,應用免疫熒光雙標法確定IL-10Rα在神經元上的定位情況。其次,利用基因干擾技術沉默神經元上的IL-10Rα基因或用JAK抑制劑(0.5或1μM)預處理神經元1 h,之后IL-10(50 ng/m L)處理1 h后再加入LPS作用8 h或24 h。應用免疫熒光細胞化學法檢測DA能神經元和非DA能神經元的數(shù)目;Western blot法檢測培養(yǎng)物中COX-2、TNF-α、BDNF、GDNF、活化的caspase-3和caspase-9的表達;ELISA方法檢測培養(yǎng)上清中TNF-α的濃度。另外,JAK抑制劑預處理的情況下,應用原位末端標記法檢測神經元凋亡率;Western blot法檢測磷酸化Stat3的水平。結果:1.中腦腹側細胞培養(yǎng)體系中,IL-10減輕LPS誘導的神經炎癥和DA能神經元缺失:LPS單獨處理培養(yǎng)物可導致DA能和非DA能神經元數(shù)目減少,i NOS、COX-2、IL-1β和TNF-α水平升高,NO和H2O2濃度增加,BDNF表達降低,但IGF-1增加,GDNF變化不明顯。IL-10預處理可明顯減輕LPS產生的毒性作用。2.中腦腹側細胞培養(yǎng)體系中,IL-10減輕MPP+誘導的神經炎癥和DA能神經元缺失:MPP+單獨處理培養(yǎng)物可導致DA能神經元數(shù)目減少,i NOS、COX-2、IL-1β和TNF-α水平升高,BDNF、IGF-1和GDNF水平降低。IL-10預處理可明顯減輕MPP+產生的上述毒性作用。3.中腦腹側神經元-小膠質細胞共培養(yǎng)體系中,IL-10減輕LPS誘導的神經炎癥和DA能神經元缺失:LPS單獨處理培養(yǎng)物可導致DA能和非DA能神經元數(shù)目減少,i NOS、COX-2、IL-1β和TNF-α水平升高,BDNF、IGF-1和GDNF水平降低。IL-10預處理可明顯減輕LPS產生的上述毒性作用。4.中腦腹側神經元-星形膠質細胞共培養(yǎng)體系中,IL-10減輕LPS誘導的炎癥介質上調:LPS單獨處理培養(yǎng)物,DA能和非DA能神經元數(shù)目變化不明顯,i NOS、COX-2、IL-1β和TNF-α水平升高,同時BDNF基因、蛋白表達和IGF-1基因表達也增加。GDNF變化不明顯,未檢測到IGF-1的蛋白表達。IL-10預處理可明顯抑制炎癥介質的生成。5.中腦腹側單純神經元培養(yǎng)體系中,IL-10減輕MPP+誘導的神經炎癥,DA能神經元凋亡和缺失:TNF-α與受損DA能神經元有共定位。MPP+單獨處理培養(yǎng)物可導致DA能神經元數(shù)目減少,TNF-α、活化的caspase-3和caspase-9水平升高。IL-10預處理可明顯減輕MPP+的上述毒性作用。中和性TNF-α抗體預處理培養(yǎng)物可降低培養(yǎng)物中促凋亡酶的表達,減輕DA能神經元缺失,但不能增強IL-10的作用。6.中腦腹側單純神經元培養(yǎng)體系中,IL-10通過與神經元上IL-10受體相互作用激活神經元內JAK-STAT3信號通路抑制LPS誘導的神經炎癥和神經元凋亡:LPS受體TLR-4與Neu N有共定位,說明LPS可通過其受體直接作用于神經元。LPS單獨處理培養(yǎng)物可導致DA能和非DA能神經元數(shù)目減少,COX-2基因表達、TNF-α水平、活化的caspase-3和caspase-9蛋白表達增加,BDNF、磷酸化的Stat3水平下降,神經元凋亡率升高,但COX-2蛋白表達和GDNF水平變化不明顯。IL-10預處理可明顯減輕LPS的上述毒性作用。未檢測到i NOS、IL-1β和IGF-1的表達。IL-10Rα與Neu N有共定位。但沉默神經元上的IL-10Rα基因或JAK抑制劑預處理神經元后,IL-10不能減輕LPS誘導的神經元缺失,也不能減輕TNF-α和促凋亡酶的上調以及BDNF的下調。另外,JAK抑制劑預處理神經元后,IL-10也不能降低神經元凋亡率,不能減輕Stat3的磷酸化水平下調。結論:1.IL-10可通過作用于膠質細胞,下調炎癥介質,上調神經營養(yǎng)因子,抑制神經炎癥和多巴胺能神經元缺失,發(fā)揮保護神經元的作用。2.IL-10也可通過與神經元上IL-10Rα相互作用激活神經元內JAK-STAT3信號通路,下調炎癥介質,上調神經營養(yǎng)因子,降低促凋亡酶水平,減少神經元凋亡,直接發(fā)揮保護神經元的作用。
[Abstract]:Objective: -10 (interleukin-10, IL-10) is one of the most important and most effective anti-inflammatory cytokines. There are many cells in the central nervous system that produce IL-10 or express IL-10 receptors. This suggests that IL-10 can regulate neuroinflammatory responses in the brain related cells. The study shows that there is a heavy weight between Parkinson's disease and neuro inflammation. In addition, IL-10 also has anti apoptosis effect, then we speculate whether IL-10 can prevent and control Parkinson's disease with its anti-inflammatory and anti apoptotic effects. Therefore, this study observed the effects of IL-10 on neuroinflammation and the loss of neurons in the neuroinflammation and dopamine (dopamine, DA) neurons in the process of PD development, and on its mechanism and signaling pathway. To clarify the protective effect of IL-10 on DA neurons in order to explore the therapeutic potential of IL-10 and provide experimental basis for the prevention and control of PD. Methods: 1. cells culture: (1) medium ventral ventral cell culture of the middle brain: cultured the ventral mesencephalon of the rat embryo for 14 + 0.5 days and culture for 7 days. (2) the simple neuron culture of the ventral ventral ventral culture of the middle brain Culture: the mesencephalic ventral tissue of the rat embryo was cultured for 14 + 0.5 days, and the cells were cultured with cytarabine to inhibit the growth of glial cells for 7 days. (3) the ventral ventral neurons of the middle brain - microglia were cultured in the middle brain. The primary cells were cultured in the cerebral cortex of the newborn rats. After 12-14 days, the microglia was added to the microglia. The neurons were trained for 6 days and 24 h later. (4) the ventral ventral neurons and astrocytes were cultured in the middle brain. The primary cultured rat cerebral cortex was cultured, and the microglia were removed for 12-14 days. At least 4 times, astrocytes were obtained and cultured for 6 days, and 24 h after 24 h. IL-10 inhibits the detection of LPS induced neuroinflammation and DA neuron loss: using IL-10 (15,50 or 150 ng/m L) preconditioning culture 1 h, and then adding LPS (50 ng/m L) action 8 h (detection of apoptosis related protein expression) or 24 applied immunofluorescence cytochemical method for detecting the number of neurons and non reactive neurons The levels of the inflammatory mediators (I NOS, COX-2, IL-1 beta and TNF- alpha) and neurotrophic factors (BDNF, IGF-1 and GDNF) in the culture were detected by ELISA method to detect the concentration of IL-1 beta, TNF- alpha or IGF-1 in the culture supernatant. In the system, the localization of LPS receptor TLR-4 on the neuron (Neu N+) was observed by immunofluorescence double labeling method. The protein expression of the apoptotic enzyme (activated caspase-3 and caspase-9) was detected by Western blot method. In situ terminal labeling method was used to detect the neuron apoptosis rate in the culture system of ventral ventral cells in the neuronal apoptosis rate, and IL-10 inhibited the MPP+ induced God. Detection of inflammation and DA neuron loss: the number of DA neurons was detected by IL-10 (15 or 50 ng/m L) pre treated culture 1 h and then MPP+ (5 mu M) used by 24 h. to detect the number of DA neurons. Western blot method was used to detect the inflammatory mediators in the culture. The expression of IL-1 beta, TNF- alpha and IGF-1 in the culture supernatant of the supernatant in the culture system of simple neuron culture in.4., IL-10 inhibits the detection of neuroinflammation and DA neuron loss induced by MPP+: pre treated with IL-10 (15 or 50 ng/m L) after 1 h, and then add (5 mu) to immunofluorescence The number of DA neurons was detected by the method of learning, and the expression of activated caspase-3 and caspase-9 in the culture was detected by the Western blot method. We conceived that IL-10 could reduce the loss of DA energy by reducing TNF- a, inhibiting apoptosis and reducing the loss of DA neurons. Therefore, the localization of TNF- alpha in the damaged DA energy neurons was determined by the double immunofluorescence method, and the Western blot method was used. The protein expression of TNF- alpha was detected and the concentration of TNF- alpha in the culture supernatant was detected by ELISA method. In order to confirm the role of TNF- alpha in MPP+ induced neuritis, we further used neutralized TNF- alpha antibody (1 mu g/m L) to pretreat the culture, plus or without IL-10 (50 ng/m L) treatment 1, and then add (5 mu) to 8 or 24 applications. The number of DA neurons was detected by immunofluorescence cytochemistry, and the Western blot method was used to detect the activation of Caspase-3 and caspase-9 in the culture medium.5. in the simple neuron culture system in the ventral ventral region of the brain. IL-10 suppressed neuro inflammation and neuron apoptosis induced by LPS through the IL-10 receptor on the neuron and the JAK-STAT3 signaling pathway in the neuron. Detection: first, the localization of IL-10R alpha on neurons was determined by immunofluorescence double labeling. Secondly, the gene interference technique was used to silence the IL-10R alpha gene on the neuron or the neuron 1 h was pretreated with JAK inhibitor (0.5 or 1 M), and then IL-10 (50 ng/m L) was treated after 1 h and then added to LPS to act 8 h or 24 h. to apply immunofluorescence cytochemistry. The number of DA neurons and non DA neurons was detected by the method. The expression of COX-2, TNF- a, BDNF, GDNF, activated caspase-3 and caspase-9 were detected by Western blot, and ELISA method was used to detect the concentration of TNF- alpha in the culture supernatant. The lot method was used to detect the level of phosphorylated Stat3. Results: in 1. midbrain ventral cell culture system, IL-10 alleviated LPS induced neuroinflammation and DA energy loss. LPS alone treated culture could lead to the decrease in the number of DA and non DA neurons, I NOS, COX-2, IL-1 beta and alpha levels increased, but the expression decreased, but increased Addition, the changes of GDNF were not obvious,.IL-10 preconditioning significantly alleviated the toxicity of LPS in the culture system of ventral ventral cells in.2., IL-10 alleviated MPP+ induced neuroinflammation and the deletion of DA neurons: MPP+ alone treated culture could lead to a decrease in the number of DA neurons, I NOS, COX-2, beta and alpha levels. The reduction of.IL-10 preconditioning significantly alleviated the above-mentioned toxic effect of MPP+ in the co culture system of ventral ventral neurons - microglia in.3., and IL-10 alleviated LPS induced neuroinflammation and DA energy loss. LPS alone treated culture could lead to a decrease in the number of DA and non DA neurons, I NOS, COX-2, beta and alpha levels increased. F, IGF-1 and GDNF levels reduced.IL-10 preconditioning significantly alleviated the above-mentioned toxic effects of LPS in the co culture system of ventral ventral neuron - astrocytes in.4., and IL-10 alleviated the up regulation of the inflammatory mediators induced by LPS: LPS alone, the number of DA energy and non DA neurons did not change obviously. At the same time, the BDNF gene, the protein expression and the expression of IGF-1 gene also increased the change of.GDNF, and the.IL-10 preconditioning of the protein expression of IGF-1 could obviously inhibit the formation of the ventral ventral neuron culture system in the inflammatory mediator, IL-10 alleviated MPP+ induced neuritis, DA neuron apoptosis and deletion: TNF- alpha and damaged DA energy. The number of DA neurons was reduced by a co location of.MPP+ in neurons, and the number of DA neurons decreased. TNF- alpha, activated caspase-3 and caspase-9 levels increased the toxicity of MPP+. Neutralized TNF- a antibody preconditioning could reduce the expression of apoptotic enzymes in the culture and reduce the loss of DA neurons. But it can not enhance the role of IL-10 in the.6. ventral ventral simple neuron culture system, IL-10 can inhibit LPS induced neuroinflammation and neuronal apoptosis by activating the JAK-STAT3 signaling pathway of IL-10 receptor on the neuron on the neuron. The LPS receptor TLR-4 and Neu N have co localization, indicating that LPS can directly act on the neurons through its receptors. LPS alone can reduce the number of DA and non DA neurons, COX-2 gene expression, TNF- alpha level, activated caspase-3 and caspase-9 protein expression, BDNF, phosphorylation Stat3 level decreased, neuron apoptosis rate increased, but COX-2 protein expression and GDNF water level changes are not obvious.IL-10 preconditioning can significantly reduce the upper level The expression of I NOS, IL-1 beta and IGF-1 expressed.IL-10R alpha with Neu N. But after the IL-10R a gene or JAK inhibitor pretreated neurons on the silent neuron, IL-10 did not reduce the LPS induced neuron loss, nor could it lessen the regulation of TNF- alpha and apoptotic enzymes and down regulation. IL-10 can not reduce the apoptosis rate of neurons and can not reduce the rate of apoptosis of Stat3. Conclusion: 1.IL-10 can be used in glial cells, down regulation of inflammatory mediators, up regulation of neurotrophic factors, inhibition of neuroinflammation and dopaminergic neuron loss, and the role of.2.IL-10 to protect neurons through neurons and neurons. The interaction of IL-10R alpha activates the JAK-STAT3 signaling pathway in the neuron, down regulate the inflammatory mediators, up-regulation the neurotrophic factors, reduce the level of apoptotic enzymes, reduce the apoptosis of neurons, and play the role of protecting neurons directly.

【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R742.5
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本文編號：1841857